Uraguchi Shimpei, Ohshiro Yuka, Okuda Mayu, Kawakami Shiho, Yoneyama Nene, Tsuchiya Yuta, Nakamura Ryosuke, Takanezawa Yasukazu, Kiyono Masako
Department of Public Health, School of Pharmacy, Kitasato University, Tokyo, Japan.
Front Plant Sci. 2022 Aug 12;13:986600. doi: 10.3389/fpls.2022.986600. eCollection 2022.
We aimed to efficiently enhance plant Hg(II) tolerance by the transgenic approach utilizing a bacterial mercury transporter MerC, an Arabidopsis mesophyll specific promoter , and a vacuolar membrane targeting syntaxin AtVAM3/SYP22. We generated two independent homozygous Arabidopsis pRBCS1A-TCV lines expressing under the control of . Quantitative RT-PCR showed that the transgene was expressed specifically in shoots of pRBCS1A-TCV lines. Confocal analyses further demonstrated the leaf mesophyll specific expression of mT-Sapphire-MerC-AtVAM3. Confocal observation of the protoplast derived from the F1 plants of the pRBCS1A-TCV line and the tonoplast marker line p35S-GFP-δTIP showed the tonoplast colocalization of mT-Sapphire-MerC-AtVAM3 and GFP-δTIP. These results clearly demonstrated that mT-Sapphire-MerC-AtVAM3 expression in Arabidopsis is spatially regulated as designed at the transcript and the membrane trafficking levels. We then examined the Hg(II) tolerance of the pRBCS1A-TCV lines as well as the p35S-driven MerC-AtVAM3 expressing line p35S-CV under the various Hg(II) stress conditions. Short-term (12 d) Hg(II) treatment indicated the enhanced Hg(II) tolerance of both pRBCS1A-TCV and p35S-CV lines. The longer (3 weeks) Hg(II) treatment highlighted the better shoot growth of the transgenic plants compared to the wild-type Col-0 and the pRBCS1A-TCV lines were more tolerant to Hg(II) stress than the p35S-CV line. These results suggest that mesophyll-specific expression of MerC-AtVAM3 is sufficient or even better to enhance the Arabidopsis Hg(II) tolerance. The Hg accumulation in roots and shoots did not differ between the wild-type Col-0 and the MerC-AtVAM3 expressing plants, suggesting that the boosted Hg(II) tolerance of the transgenic lines would be attributed to vacuolar Hg-sequestration by the tonoplast-localized MerC. Further perspectives of the MerC-based plant engineering are also discussed.
我们旨在通过转基因方法，利用细菌汞转运蛋白MerC、拟南芥叶肉特异性启动子和液泡膜靶向 syntaxin AtVAM3/SYP22，有效提高植物对Hg(II)的耐受性。我们构建了两个独立的纯合拟南芥pRBCS1A-TCV株系，它们在[具体启动子名称未给出]的控制下表达。定量逆转录聚合酶链反应（qRT-PCR）表明，转基因在pRBCS1A-TCV株系的地上部特异性表达。共聚焦分析进一步证明了mT-Sapphire-MerC-AtVAM3在叶肉中的特异性表达。对pRBCS1A-TCV株系的F1植株原生质体与液泡膜标记株系p35S-GFP-δTIP进行共聚焦观察，结果显示mT-Sapphire-MerC-AtVAM3与GFP-δTIP在液泡膜上共定位。这些结果清楚地表明，拟南芥中mT-Sapphire-MerC-AtVAM3的表达在转录水平和膜运输水平上均按设计进行空间调控。然后，我们在各种Hg(II)胁迫条件下，检测了pRBCS1A-TCV株系以及p35S驱动的MerC-AtVAM3表达株系p35S-CV对Hg(II)的耐受性。短期（12天）Hg(II)处理表明，pRBCS1A-TCV和p35S-CV株系对Hg(II)的耐受性均增强。较长时间（3周）的Hg(II)处理突出显示，与野生型Col-0相比，转基因植物地上部生长更好，且pRBCS1A-TCV株系比p35S-CV株系对Hg(II)胁迫更具耐受性。这些结果表明，MerC-AtVAM3在叶肉中的特异性表达足以甚至更有利于提高拟南芥对Hg(II)的耐受性。野生型Col-0与表达MerC-AtVAM3的植株在根和地上部的Hg积累没有差异，这表明转基因株系对Hg(II)耐受性的提高归因于液泡膜定位的MerC对Hg的区隔化作用。文中还讨论了基于MerC的植物工程的进一步前景。
